MEDICATION AND DIAGNOSTIC KIT FOR INHIBITING METASTASIS AND INVASION OF BREAST CANCER, shRNA MOLECULE FOR SILENCING EXPRESSION OF HUMAN LINC01614 AND APPLICATION THEREOF

ABSTRACT

The invention provides a medication and a diagnostic kit for inhibiting metastasis and invasion of breast cancer, an shRNA molecule for silencing expression of human LINC01614 and applications thereof. The shRNAs obtained by the invention can interfere with the expression of LINC01614, thereby reducing the migration and invasion ability of tumor cells, inhibiting the expression of EMT proteins, and inhibiting tumor formation and lung metastasis in an animal model in vivo. The invention provides a new solution for targeted therapy of breast cancer. Therefore, the kit for diagnosing metastasis and invasion of breast cancer and the medication for treating metastasis and invasion of breast cancer are developed. The invention provides a new way and strategy for diagnosing and treating metastasis and invasion of breast cancer.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority benefit of China application serialno. 202111494647.0, filed on Dec. 8, 2021. The entirety of theabove-mentioned patent application is hereby incorporated by referenceherein and made a part of this specification.

BACKGROUND Technical Field

The present invention belongs to the technical field of biomedicine andmainly belongs to a medication and a diagnostic kit for inhibiting themetastasis and invasion of breast cancer.

Description of Related Art

Breast cancer is a common malignant tumor and ranks the first in femalecancers due to its high incidence rate. In recent years, the femalebreast cancer incidence rate in China rises yearly among the younger agegroup. The discovery and development of anti-tumor treatment methodsimprove tumor patients' survival rate and life quality. However,patients receiving tumor drug treatment usually generate drugresistance, which leads to reduced curative effect, tumor recurrence andmetastasis, and finally treatment failure. Invasion and metastasis ofbreast cancer are multi-factor and multi-step complex processes. Theyare regulated and controlled by the expression of multiple genes.However, the specific molecular mechanism is not completely clear atpresent.

LncRNAs are kind of functional RNA molecules with a length of more than200 nt and couldn't encode proteins. It participates in regulating andcontrolling various biological processes in cells. At present, thousandsof lncRNAs participate in regulating and controlling the expression ofgenes. Much research reports the abnormal expression=of lncRNAs invarious malignant tumors. The influence of the abnormal expression ofLncRNAs in human disease is more and more prominent. LINC01614 is a newtumor diagnosis and prognosis index. Our studies found that highexpression of LINC01614 is positively correlated with multiple genes ina cell adhesion signal channel. It is also correlated withEpithelial-Mesenchymal Transition (EMT), which affects the metastasisand prognosis in various cancers. Public database mining suggested thatLINC01614 is highly expressed in multiple cancers, including breastcancer, non-small cell lung cancer, gastric cancer, glioma, etc., whichprompts the expression of LINC01614 is closely correlated with theoccurrence and development of tumors. However, there are not manyreports on LINC01614 in the literature so far.

RNA interference (RNAi) is a double-stranded RNA that mediatessequence-specific gene knockdown and widely exists in living organisms.It can be generally used in gene function exploration, infectiousdisease treatment, and malignant tumor treatment due to the ability tospecifically knock down the expression of specific genes. The shorthairpin RNA (shRNA) is a section of exogenous RNA sequence with astem-loop structure. It can be processed into siRNAs in cells. The siRNAcombined with proteins and formed an RNA-induced knockdown complex(RISC) and combined with homologous mRNAs, resulting in mRNAdegradation. The shRNAs have strict targeting properties. The selectionof specific target sites must match the correct position. Theinterference efficiency of different target sites on the same gene wouldbe different.

In the present invention, LINC01614 specific silencing shRNAs areconstructed. After transfected the shRNAs into tumor cells, themetastasis and invasion of the tumor cells were effectively inhibited,and the expression of EMT-related proteins of the tumor cells wereinhibited. It provides an application basis for further in-depthexploration of the molecular mechanism of LINC01614 in breast cancermetastasis and invasion.

SUMMARY

The objective of the present invention is to design and provide atechnical solution of a medication and a diagnostic kit for inhibitingmetastasis and invasion of breast cancer. To achieve the aboveobjective, the present invention adopts the following technicalsolution:

First, the present invention provides a kit for inhibiting metastasisand invasion of breast cancer. The kit comprises an agent for detectingan LINC01614 inhibition effect in human breast cancer cells, and theagent consists of primers shown as SEQ ID No. 10-13.

Second, the present invention provides a medication for inhibitingmetastasis and invasion of breast cancer. The medication comprisesshRNAs for inhibiting expression of LINC01614, wherein the shRNAs forinhibiting expression of LINC01614 comprises an shRNA-1 and/or anshRNA-2. A sense strand nucleotide sequence of the shRNA-1 is shown asSEQ ID No. 3. An antisense strand nucleotide sequence of the shRNA-1 isshown as SEQ ID No. 4. A sense strand nucleotide sequence of the shRNA-2is shown as SEQ ID No.5. An antisense strand nucleotide sequence of theshRNA-2 is shown as SEQ ID No. 6.

Further, the medication is recombinant lentivirus expression vectors orrecombinant lentiviruses that expresses the shRNAs, which could inhibitthe expression of LINC01614.

Additionally, the present invention provides shRNAs for silencing theexpression of LINC01614, which comprises an shRNA-1 and/or an shRNA-2. Asense strand nucleotide sequence of the shRNA-1 is shown as SEQ ID No.3. An antisense strand nucleotide sequence of the shRNA-1 is shown asSEQ ID No. 4. A sense strand nucleotide sequence of the shRNA-2 is shownas SEQ ID No. 5. An antisense strand nucleotide sequence of the shRNA-2is shown as SEQ ID No. 6.

Furthermore, the present invention provides an application of shRNAs forsilencing expression of LINC01614 in preparation of a medication forinhibiting the expression of LINC01614.

Moreover, the present invention provides an application of shRNAs forsilencing expression of LINC01614 in preparation of a medication forinhibiting metastasis and invasion of human breast cancer cells,MDA-MB-231 and Hs578T.

Finally, the present invention provides an application of shRNAs forsilencing expression of LINC01614 in preparation of a medication forinhibiting metastasis and invasion of breast cancer.

The present invention has the following beneficial effects:

1, it explicitly reduces the expression of LINC01614. And it inhibitsthe tumor formation, metastasis and invasion ability of breast cancer,resulting in controlling breast cancer metastasis. And

2, the shRNAs are constructed on lentiviral vectors. The gene silencingis performed by transfecting breast cancer cell strains, MDA-MB-231 andHs578T, with shRNA-lentivirus. The efficiency of gene silencing isimproved. And

3, therefore, the kit for diagnosing metastasis and invasion of breastcancer and the medication for treating metastasis and invasion of breastcancer are developed, and the present invention provides a new way andstrategy for the diagnosis and treatment of metastasis and invasion ofbreast cancer.

The shRNAs in the present invention are not reported yet. The shRNAsmentioned above can reduce the expression of LINC01614, reduce migrationand invasion ability of tumor cells, inhibit the expression of EMTrelated protein, and inhibit tumor formation and lung metastasis invivo. The present invention provides a new solution for targeted therapyof breast cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a validation effect of lentivirus-mediated shRNAs onLINC01614 silencing in human breast cancer cells, MDA-MB-231 and Hs578Tcells. * represents that compared with sh-Control, *P is less than 0.05,**P is less than 0.01, and ***P is less than 0.001.

FIG. 2 shows the clone formation of MDA-MB-231 and Hs578T cells thattransfected with lentivirus.

FIG. 3 shows the migration and invasion ability of MDA-MB-231 and Hs578Tcells that transfected with lentivirus.

FIG. 4 shows the protein expression of EMT-related proteins, E-cadherinand N-cadherin, in MDA-MB-231 and Hs578T cells that transfected withlentivirus.

FIG. 5 shows subcutaneous tumor formation modelling of nude mice withMDA-MB-231 transfected with lentivirus to knockdown LINC01614. Tumorvolume and weight were detected.

FIG. 6 shows the breast cancer lung metastasis model by tail veininjection with MDA-MB-231 transfected with lentivirus. Lung metastasiswas detected.

DESCRIPTION OF THE EMBODIMENTS

The following example illustrates the present invention but are notintended to limit the scope of the present invention. If notspecifically indicated, the technical means used in the examples areconventional means well known to those skilled in the art.

Statistical Analysis

Data was subjected to statistical analysis by using SPSS 22.0 software.Measurement data was represented by mean±standard deviation. One-wayANOVA and t-test were used to examine the difference from one anotheramong groups, and p-value less than 0.05 indicated a significantdifference.

Example 1: Design of LINC01614 Specific shRNAs

The shRNA sequences were listed below:

shRNA-1: (shown as SEQ ID No. 1) TTCCTTAAAGTAGCAATCTTAGC; shRNA-2:(shown as SEQ ID No. 2) GACAAGTTCAGTGGAAACTTTCT.

A sense strand nucleotide sequence of the shRNA-1:

(shown as SEQ ID No. 3)5′-CCGGTTCCTTAAAGTAGCAATCTTAGCCTCGAGGCTAAGATTGCTA CTTTAAGGAATTTTTG-3′;

An antisense strand nucleotide sequence of the shRNA-1:

(shown as SEQ ID No. 4)5′-AATTCAAAAATTCCTTAAAGTAGCAATCTTAGCCTCGAGGCTAAGA TTGCTACTTTAAGGAA-3′.

A sense strand nucleotide sequence of the shRNA-2:

(shown as SEQ ID No. 5)5′-CCGGGACAAGTTCAGTGGAAACTTTCTCTCGAGAGAAAGTTTCCAC TGAACTTGTCTTTTTG-3′;

An antisense strand nucleotide sequence of the shRNA-2:

(shown as SEQ ID No. 6)5′-AATTCAAAAAGACAAGTTCAGTGGAAACTTTCTCTCGAGAGAAAGT TTCCACTGAACTTGTC-3′;sh-LINC01614-1: (shown as SEQ ID No. 7)CCGGTTCCTTAAAGTAGCAATCTTAGCCTCGAGGCTAAGATTGCTACTT TAAGGAATTTTTG;sh-LINC01614-2: (shown as SEQ ID No. 8)CCGGGACAAGTTCAGTGGAAACTTTCTCTCGAGAGAAAGTTTCCACTGA ACTTGTCTTTTTG.sh-Control: (shown as SEQ ID No. 9) TTCTCCGAACGTGTCACGT.

Example 2: Construction of shRNA-Lentiviral Expression Vectors

The ahU6-MCS-CBh-gcGFP-IRES-puromycin vector was digested by usingrestriction enzymes EcoR and Age I. Digest products were separated bygel electrophoresis. Target segments were purified. Resuspending andannealing both sense and antisense shRNA oligonucleotides with the ratioof 1:1. The annealed shRNA oligonucleotides were inserted between therestriction sites in vector by ligation. Ligation reaction wastransformed into competent Escherichia coli DH5a. Candidate colonieswere isolated and amplified. the shRNA inserts of the target vectorswere identified by sequencing and alignment.

Table 1 Designed specific primer based on shRNA inserts NO. 5′ STEM LoopSTEM 3′ LINC0161 Ccgg TTCCTTAAAGTAGC CTC GCTAAGATTGCT TTTTTg 4-1-sh1-forAATCTTAGC GAG ACTTTAAGGAA LINC0161 aattcaaa TTCCTTAAAGTAGC CTCGCTAAGATTGCT 4-1-sh1-rev aa AATCTTAGC GAG ACTTTAAGGAA LINC0161 CcggGACAAGTTCAGTGG CTC AGAAAGTTTCCA TTTTTg 4-1-sh2-for AAACTTTCT GAGCTGAACTTGTC LINC0161 aattcaaa GACAAGTTCAGTGG CTC AGAAAGTTTCCA4-1-sh2-for aa AAACTTTCT GAG CTGAACTTGTC

Specific steps are as follows:

(1) Linearized and purified of the vectors. The enzyme digestion systemis shown in Table 2:

TABLE 2 Enzyme digestion system Agent Volume (μl) ddH₂O 41 10 × CutSmartBuffer² 5 Purified plasmid DNA (1 μg/μL) 2 Age I (10 U/μl) 1 EcoR I (10U/μl) 1 Total 50

After incubating for 1 h at 37° C. (optimum temperature), reactions wereseparated by electrophoresis in a 1% agarose gel. Target segments werepurified and subjected to enzyme digestion.

(2) For ligation, linearization vector and annealed shRNAoligonucleotides were ligated by T4 DNA ligase at 16° C. for 1-3 h orovernight. The ligating reaction is shown in Table 3:

TABLE 3 Ligating reaction Agent Volume (μl) Linearized vector (100ng/μL)  1* Double-stranded DNA (shRNA) (100 ng/μL) 1 10 × T4 DNA ligasebuffer 2 T4 DNA ligase 1 dd H₂O Top up to 20 *Corresponding adjustmentsshould be made according to the size of the vector.

(3) PCR Identification and Sequencing of Positive Transformed Colonies

Transform 50 μl competent E. coli DH5a cells with 50 of the ligationreaction. Leave the mixture on ice for 30 min. Heat shocks the mixturein the 42° C. bath for 90 sec then immediately incubated the mixture inthe ice for 3 min. Add 950 μL of antibiotic-free liquid LB medium to thecells and incubated in 37° C. shaking incubator for 45 min with a speedof 150 rpm. Add and spread 150 μl cells suspension from eachtransformation on LB agar+ampicillin (100 μg/ml) plates. Incubate platesat 37° C. overnight. Pick 10 candidate isolated colonies from eachtransformation and inoculate each into LB+ampicillin (50-100 μg/ml).Grow overnight at 37° C. with shaking (150 RPM). The cultures werecollected, and plasmid vectors were extracted.

shRNA inserts were identified by sequencing and alignment. Correctaligned vectors were identified as a successfully constructed shRNAlentiviral expression vectors.

(4) Lentivirus Packaging and Titer Determination

The 293T cells were co-transfected with extracted lentivirus expressionvectors and packaging plasmids (Shanghai Genechem Co., Ltd., provided atransfection agent). Harvest and filter the lentiviral supernatantsthrough a 0.45 μm low protein binding filter to remove cellular debriswithin 48 h. The concentrated viruses were stored at −80° C.

According to the examples, three recombinants lentivirus,sh-LINC01614-1, sh-LINC01614-2 and sh-Control were obtained.

Example 3: Transfected Human Breast Cancer Cells, MDA-MB-231 and Hs578TCells, with Sh-LINC01614-1, Sh-LINC01614-2 and Sh-Control Lentivirus

MDA-MB-231 and Hs578T cells were prepared and inoculated to a 6-poreplate with a density of 2.5×10⁵ cells/pore. When the cells densityreached 70%, add sh-LINC01614-1, sh-LINC01614-2 or sh-Control lentivirus(MOI=10) supernatant to the cell and transduce for 24 hour. Remove anddiscard the lentivirus-containing transduction medium and replace withfresh growth medium. Continue to incubate the cells for 72 hours. After72 h, the GFP expression was analysis by a fluorescence microscope. Whenthe expression reached more than 90% indicated a successfultransfection.

Example 4: Detect the Knockdown Efficiency of LINC01614 in TransfectedHuman Breast Cancer Cells, MDA-MB-231 and Hs578T

MDA-MB-231 and Hs578T cells that transfected with sh-LINC01614-1,sh-LINC01614-2 or sh-Control lentivirus were harvested. The total RNA ofeach group was extracted with Total RNA extraction kit (15596026,Invitrogen Company) according to the manufacturer's instructions. ThecDNAs were synthesized by reverse transcription with a reversetranscription kit (RR037A, Takara Company). The Real-time PCR detectionof LINC01614 (amplification primers shown in Table 4) was performedusing SYBR® Select Master Mix (Applied Biosystems, cat: 4472908) on ABI7900 system (Applied Biosystems, Foster City, Calif., USA) with theprimers (Table 4) according to the manufacturer's instructions. Theprocedure was showed as follows: initial denaturation for 2 minutes at94° C.; 40 cycles of denature 30 s at 94° C., anneal primers for 30 s at55° C. and extend DNA for 1 min at 72° C. The Real-Time PCR adopted a2^(−ΔΔCt) method for relative quantitative analysis.

TABLE 4 Real-time PCR Primer sequences Gene Primer sequence LINC01614F5′-AACCAAGAGCGAAGCCAAGA-3′ (shown as SEQ ID No. 10) LINC01614R5′-GCTTGGACACAGACCCTAGC-3′ (shown as SEQ ID No. 11) GAPDH F5′-CTGGTAAAGTGGATATTGTTGCCAT-3′ (shown as SEQ ID No. 12) GAPDH R5′-TGGAATCATATTGGAACATGTAAACC-3′ (shown as SEQ ID No. 13)

The results showed that the expression of LINC01614 in sh-LINC01614-1group and sh-LINC01614-2 group were significantly downregulated thanthat of sh-Control group (P<0.001), and the silencing effect ofsh-LINC01614-2 is better (shown in FIG. 1 ). Thus, the primers inExample 4 could be used as a kit for detecting the expression ofLINC01614 in human breast cancer cells.

Example 5: Knocking Down of LINC01614 Inhibited the Clone Formation ofMDA-MB-231 and Hs578T Cells

MDA-MB-231 and Hs578T cells were plated in six-well plates overnight andthen transfected with sh-LINC01614-1, sh-LINC01614-2 or sh-Controllentivirus. After the removal of the drug-containing medium, the cellswere washed using PBS, trypsinized and plated at a low density (2000cells/well in six-well plates). The cells were cultivated for 2-3 weeksin a humidified atmosphere containing 5% CO2 at 37° C. and the mediumwas refreshed every two days. Then, cells were washed with PBS for twiceand fixed with 4% of paraformaldehyde for 30 minutes at room temperatureand then washed with 1×PBS. Cells were stained with 0.5% crystal violet(Sigma Chemical Co, St. Louis, Mo.) at room temperature. The excess dyewas removed by washed 3 times with distilled water. The cell coloniescontaining more than 50 cells were counted under microscope. Eachexperiment was independently repeated for 3 times.

As shown in FIG. 2 , the clone formation of cells that transfected bysh-LINC01614-1 and sh-LINC01614-2 were significantly reduced than thatof sh-Control (P<0.01).

Example 6: Knocking Down of LINC01614 Inhibited Migration and Invasionof MDA-MB-231 and Hs578T Cells

MDA-MB-231 and Hs578T cells infected with sh-LINC01614-1, sh-LINC01614-2or sh-Control lentivirus were serum starved for 12 h and then digestedwith trypsin, respectively. Cells were centrifuged, diluted andresuspended with serum-free medium. For migration analysis, thetranswell insert membrane is coated with Matrigel. A total of 30 μldiluted Matrigel was added to the upper chamber of each Transwellinsert, which was then incubated at 37° C. for 120 min. The upperchamber of the Transwell incubated with 100 μl cells (2×10⁴cells/chamber) suspended in serum-free medium. The lower chamber wassupplemented with 500 μl medium containing 20% FBS and incubated for 24h at 37° C. Cells on the upper surface of the membrane were removedusing a cotton swab. Cells on the bottom surface of insert membrane werefixed with 4% of paraformaldehyde for 30 minutes at room temperature andsubsequently stained with 0.1% crystal violet for 30 min at roomtemperature. Migration cells were photographed and counted in fiverandom fields of view under a microscope. For invasion analysis, thetranswell insert membrane is NOT coated with Matrigel. The procedure isidentic with the migration analysis, the only difference is WITHOUT thepresence of Matrigel. Each experiment was independently repeated for 3times.

As shown in FIG. 3 , the migration and invasion ability ofsh-LINC01614-1 group and sh-LINC01614-2 group were significantly reducedcompared with the sh-Control group (P<0.05).

Example 7: Knocking Down of LINC01614 Inhibited Epithelial-MesenchymalTransition (EMT) of MDA-MB-231 and Hs578T Cells

MDA-MB-231 and Hs578T cells infected with sh-LINC01614-1, sh-LINC01614-2or sh-Control lentivirus. Total protein of 2×10⁶ cells of each groupwere extracted using Total Protein Extraction kits (KeyGen Biotech,Nanjing, China) following the manufacturer's protocol. The proteinconcentration was determined by a BSA method. Equal amount of loadingbuffer was added and denatured at 100° C. for 10 min. 20 μg of denaturedsamples were loaded and separated by 12% SDS-PAGE and transferred onto apolyvinylidene difluoride (PVDF) membrane. After blocking with 5% ofskimmed milk at room temperature for 1.5 h, the membrane was incubatedwith primary anti-rabbit E-cadherin (1: 1000, CST Company) andN-cadherin (1: 1000, CST Company) and rabbit anti-β-actin monoclonalantibody (1: 5000, Abeam Company) at 4° C. overnight, respectively. Washthe membrane in TBST for three times, 5 min each. Then, HRP-labeled goatanti-rabbit IgG (1: 5000, Abeam Company) was added and incubated at roomtemperature for 1 h. Proteins were visualized with an enhancedchemiluminescence reagent (Wanleibio Co., Ltd.) and a ChemiluminescenceDetection System (Image Lab version 5.1; Bio-Rad Laboratories, Inc.).The β-ACTIN was served as control. Each experiment was independentlyrepeated for 3 times.

The results of western blot indicated that after knocking down ofLINC01614, the expression of the EMT-related protein, E-cadherin, wasincreased, and the expression of the N-cadherin was reduced. Thesesuggested that the knocking down LINC01614 could inhibit the formationof tumor cell EMT (shown as FIG. 4 ).

Example 8: Knocking Down of LINC01614 Inhibited the Xenograft TumorSizes and Lung Metastasis of MDA-MB-231 Cells

For tumor xenograft mouse model, MDA-MB-231 cells transfected withsh-Control, sh-LINC01614-1, and sh-LINC01614-2 lentivirus. The BALB/cnude female mice were purchased from The Model Animal Research Center ofNanjing University (Nanjing, China). The mice were divided into threegroups: sh-LINC01614-1 group, sh-LINC01614-2 group and sh-Control group.Subsequently, each 5-week-old mouse was subcutaneously injected with1×10⁷ transfected MDA-MB-231 cells that suspended in 200 μl of PBSaccording to the group. The tumor sizes were monitored every 4 day. Bodyweight of mice also be monitored 20 days post injection. After 20 days,the nude mice were sacrificed by cervical dislocation. The long diameter(L) and the short diameter (W) of the tumor were measured. The tumorvolume was calculated following the formula: (V=L×W2/2. Tumor tissuealso been imaged.

For experimental lung metastasis, MDA-MB-231 cells were transinfectedwith sh-Control, sh-LINC01614-1, and sh-LINC01614-2 lentivirus.Transfected cells were injected into the 4- to 6-week-old BALB/c nudemale mice through tail veins (5×10⁶ cells/mouse). After 4 weeks, thelung tissues were dissected and fixed in 10% formalin. Metastatic fociwere counted and imaged.

As displayed in FIG. 5 , the xenograft tumor sizes of thesh-LINC01614-1, and sh-LINC01614-2 group were significantly smaller thanthat of sh-Control group. As showed in FIG. 6 , lung metastasis of thesh-LINC01614-1, and sh-LINC01614-2 group were reduced compared with thatof sh-Control. The results indicated that downregulated of LINC01614could significantly inhibit the MDA-MB-231 cell growth and lungmetastasis in vivo.

The results of Examples 6-8 showed downregulated the expression ofLINC01614 by transfection with sh-LINC01614-1, and sh-LINC01614-2lentivirus in the present invention could be used as the medication forinhibiting metastasis and invasion of breast cancer.

The preferred examples of the present invention have been specificallydescribed above. However, the present invention is not limited to theseexamples, and those skilled in the art can make various equivalentswithout departing from the spirit of the present invention.

Modifications or substitutions of these equivalent modifications orsubstitutions are all included within the scope defined by the claims ofthe present application.

What is claimed is:
 1. A kit for diagnosing metastasis and invasion ofbreast cancer, comprising an agent for detecting an LINC01614 inhibitioneffect in human breast cancer cells, wherein the agent comprises primersshown as SEQ ID No. 10-13.
 2. A medication for inhibiting metastasis andinvasion of breast cancer, comprising shRNAs for inhibiting expressionof human LINC01614, wherein the shRNAs for inhibiting expression ofhuman LINC01614 comprises a shRNA-1 and/or a shRNA-2, a sense strandnucleotide sequence of the shRNA-1 is shown as SEQ ID No. 3, anantisense strand nucleotide sequence of the shRNA-1 is shown as SEQ IDNo. 4, a sense strand nucleotide sequence of the shRNA-2 is shown as SEQID No.5, and an antisense strand nucleotide sequence of the shRNA-2 isshown as SEQ ID No.
 6. 3. The medication for inhibiting metastasis andinvasion of breast cancer according to claim 2, wherein the medicationis a recombinant lentivirus expression vector of the shRNAs forinhibiting the expression of human LINC01614 or a recombinant lentivirusof the shRNAs for inhibiting the expression of human LINC01614.
 4. AnshRNA molecule for silencing expression of human LINC01614, comprisingan shRNA-1 and/or an shRNA-2, wherein a sense strand nucleotide sequenceof the shRNA-1 is shown as SEQ ID No. 3, an antisense strand nucleotidesequence of the shRNA-1 is shown as SEQ ID No. 4, a sense strandnucleotide sequence of the shRNA-2 is shown as SEQ ID No. 5, and anantisense strand nucleotide sequence of the shRNA-2 is shown as SEQ IDNo.
 6. 5. An application of the shRNA molecule for silencing theexpression of human LINC01614 according to claim 4 in preparation of amedication for inhibiting expression of LINC01614.
 6. An application ofthe shRNA molecule for silencing the expression of human LINC01614according to claim 4 in preparation of a medication for inhibitingmetastasis and invasion of human breast cancer cells MDA-MB-231 andHs578T.
 7. An application of the shRNA molecule for silencing theexpression of human LINC01614 according to claim 4 in preparation of amedication for inhibiting metastasis and invasion of breast cancer cell.